Heterogeneous nuclear ribonucleoproteins (hnRNPs) are an abundant group of proteins which occur in the eukaryotic cell nucleus. These proteins associate with RNA polymerase II transcripts (hnRNA) to form hnRNP-complexes. The function of hnRNPs is believed to include participation in RNA processing. Originally, three groups of hnRNP proteins were isolated: the A group (A1 and A2), the B group (B1 and B2) and the C group (C1 and C2). Classically, these three groups of molecules are considered to be core proteins. At least twenty different hnRNPs have been isolated. The core protein hnRNP A1 protein is reported to facilitate RNA-RNA annealing.
Pontius, B. W. and P. Berg, Proc. Natl. Acad. Sci. U.S.A. (November 1990) 87:8403-8407, report that purified hnRNP A1 protein was found to promote the rapid renaturation of nucleic acid strands. It is reported that at 60.degree. C., the A1-mediated renaturation rate was about 300-fold greater than protein-free reactions carried out at 68.degree. C. in 1M NaCl. It is suggested that A1 may be useful in procedures that rely on nucleic acid renaturation.
Kumar, A. and S. H. Wilson, Biochemistry (December 1990) 29(48):10717-10722, report that hnRNP A1 is a major core protein of the mammalian hnRNP complex and that both intact hnRNP A1 protein and a C-terminal domain fragment are capable of potent strand-annealing activity for complementary base pairs of single-stranded RNA and DNA polynucleotides.
Munroe, S. H. and X. Dong, (February 1992) Proc. Natl. Acad. Sci. USA 89:895-899, report that the presence of hnRNP A1 protein increases the rate of annealing of complementary RNA strands by &gt;300-fold under a wide range of salt concentrations and temperatures. The maximal annealing rates are reported to be under saturating or near saturating concentrations of protein; it is also reported that annealing decreases sharply at both higher and lower concentrations of A1. It is reported that the N-terminal two-thirds of A1 displayed very limited annealing activity while the 48 amino acid residues from the glycine-rich C-terminal region promoted annealing at a level of almost one-quarter of that which is observed with intact A1.
Pontius, B. W. and P. Berg, J. Biol. Chem. (July 1992) 267(20):13815-13818, report that hnRNP A1 protein affects both the equilibrium and kinetic properties of the reaction in which complementary strands of nucleic acids renature in vitro.
There are many instances where it is desirable to promote and accelerate the process of annealing complementary strands of nucleic acid sequences. For example, it is desirable to promote annealing of PCR primers in Polymerase Chain Reaction protocols. Likewise, other examples of methods in which it is desirable to promote annealing of complementary nucleic acid sequences include, but are not limited to: Southern blot hybridizations, Northern blot hybridizations, oligonucleotide and gene fragment probe hybridization of extracted RNA or DNA or colony-lift DNA, library screening hybridizations with oligonucleotides or gene fragments, subtractive hybridization, RNase protection, or primer extension assays or any technique involving primer hybridization such as DNA or RNA sequencing.
Although hnRNP protein A1 promote more efficient nucleic acid annealing, there is need for more potent and effective reagents to promote and accelerate a complementary nucleic acid sequence annealing. The applications for such reagents are numerous and the benefits of promoting and accelerating complementary nucleic acid sequence annealing provide many advantages such as, for example, enabling more rapid diagnostic testing methods as well as less time consuming laboratory procedures.
The present invention provides a method of promoting complementary nucleic acid sequence annealing using the hnRNP U protein. The hnRNP U protein is an hnRNP which promotes annealing much more efficiently than that which is accomplished using hnRNP A1. By promoting the annealing of complementary nucleic acid sequences in a method which includes providing hnRNP U protein, the annealing of complementary nucleic strands occurs a much more efficiently. In addition, the present invention provides nucleic acid sequence which encode hnRNP U protein. The nucleic acid sequence which encode hnRNP U protein may be used to produce the protein by recombinant DNA methodology. Accordingly, large quantities of essentially pure hnRNP U protein may be produced in a rapid and efficient manner.